Prevalence, antimicrobial resistance, virulence gene profile and molecular typing of Campylobacter species isolated from poultry meat samples

Abstract Background Campylobacter jejuni and Campylobacter coli are the most significant Campylobacter species responsible for severe gastrointestinal disorders. Raw poultry meat is considered a source of Campylobacter transmission to the human population. Objectives The present study was aimed to assess the prevalence rate, antibiotic resistance properties, virulence characters and molecular typing of C. jejuni and C. coli strains isolated from raw poultry meat samples. Methods Three hundred and eighty raw poultry meat samples were collected and analysed for the presence of Campylobacter spp. using the microbial culture. Species identification was done using the Polymerase Chain Reaction. Disk diffusion was developed to assess the antimicrobial resistance pattern of isolates. The distribution of virulence and antimicrobial resistance genes was determined by PCR. Enterobacterial Repetitive Intergenic Consensus‐PCR was used for molecular typing. Results Campylobacter species were isolated from 6.25% of examined samples. C. jejuni and C. coli contamination rates were found to be 57.44% and 48.14%, respectively. C. jejuni strains harboured the highest resistance rate against serythromycin (42.59%), ampicillin (38.88%), ciprofloxacin (33.33%), chloramphenicol (31.48%) and tetracycline (31.48%). C. coli isolates harboured the highest resistance rate against ampicillin (73.07%), ciprofloxacin (73.07%), erythromycin (65.38%) and chloramphenicol (50%). AadE1 (44.44%), blaOXA‐61 (42.59%) and tet(O) (35.18%) were the most commonly detected resistance genes in C. jejuni and cmeB (34.61%) and blaOXA‐61 (34.61%) were the most commonly detected among C. coli strains. The most frequent virulence factors among the C. jejuni isolates were flaA (100%), ciaB (100%), racR (83.33%), dnaJ (81.48%), cdtB (81.48%), cdtC (79.62%) and cadF (74.07%). The most frequent virulence factors among the C. coli isolates were flaA (100%), ciaB (100%), pldA (65.38%) and cadF (61.53%). Conclusions The majority of C. jejuni and C. coli strains had more than 80% similarities in their ERIC‐PCR pattern, which may show their common source of transmission. The role of goose and quebec meat samples as reservoirs of virulent and antimicrobial resistant Campylobacter spp. was determined.

The role of goose and quebec meat samples as reservoirs of virulent and antimicrobial resistant Campylobacter spp. was determined. Campylobacter jejuni (C. jejuni) and C. coli are major species responsible for severe cases of human gastroenteritis (Igwaran & Okoh, 2019). Humans most often become infected by ingesting contaminated food, particularly undercooked poultry meat (Myintzaw et al., 2022). Poultry carcasses are typically contaminated during defeathering and evisceration by faeces leakage containing campylobacters from the cloaca (Hakeem & Lu, 2021). In most cases, campylobacteriosis is typically self-limiting; however, complications may occur in some persons. Around 1 in 1000 infected individuals develops Guillain-Barré syndrome (GBS), a thoughtful autoimmune-mediated neurological disorder that causes weakness of extremities, complete paralysis, respiratory insufficiency and death (Scallan Walter et al., 2020).
Diverse Polymerase Chain Reaction (PCR)-based typing, such as PCR sequencing, PCR-ribotyping and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) have been developed for the molecular typing of Campylobacter spp. (Igwaran & Okoh, 2020a). ERIC-PCR technique is a simple tool used to differentiate bacteria strains isolated from diverse sources. This technique is a strong tool for the exploration of prokaryotic genomes and has been reported to have improved reproducibility and high discriminatory power (Bilung et al., 2018). Its application for successful typing of Campylobacter spp. has been reported in a previous survey (Staji et al., 2018).
Data about the epidemiology of foodborne campylobacteriosis are scarce in Iran. Additionally, the exact prevalence rate, virulence characters and antimicrobial resistance of Campylobacter spp. were not well defined among poultry in Iran. Thus, the present survey was done to assess the prevalence rate, antimicrobial resistance pattern, distribution of virulence genes and the molecular typing of C. coli and C. jejuni strains isolated from poultry meat samples.
Raw poultry meat samples (100 g) were collected from the thigh muscle using sterile plastic bags. Samples were transferred in refrigerated containers at 4 • C. Samples transportation and processing were done within 2 h after collection. A colony from each medium was subjected to biochemical examinations, including Gram-staining, catalase production (3% H2O2), hippurate oxidase and hydrolysis, indoxyl acetate hydrolysis, urease activity and resistance against cephalothin (Nachamkin, 2003). Campylobacter species identification was done using the PCR (Denis et al., 1999). Suspected Campylobacter isolates were sub-cultured on Bolton broth and incubated for 48 h at 42 • C in a microaerobic condition.

Bacterial isolation and identification
According to the manufacturer's instructions, the genomic DNA was extracted from the isolates using the DNA extraction kit (Thermo   Table 1 shows the PCR conditions met to detect antimicrobial resistance genes and virulence factors (Datta et al., 2003;Obeng et al., 2012). A programmable DNA thermocycler (Eppendorf Mastercycler 5330, Eppendorf-Nethel-Hinz GmbH, Hamburg, Germany) was used in all PCR reactions. In addition, amplified samples were analysed by electrophoresis (120 V/208 mA) in a 2.5% agarose gel stained with 0.1% ethidium bromide (0.4 μg/ml). Besides, UVI doc gel documentation systems (Grade GB004, Jencons PLC, London, UK) were used to analyse images.

ERIC-PCR molecular typing
C. jejuni and C. coli isolates of different raw poultry meat samples were subjected to PCR using the ERIC primer set R1: ATGAAGCTC-CTGGGGATTCAC and R2: AAGTAAGTGACTGGGGTGAGCG (Zorman et al., 2006). The PCR reactions were verified by resolving them in 3% agarose gel in a 5× TBE buffer, stained with ethidium bromide at 90 volts for 240 min and viewed. ERIC-PCR DNA fingerprints were analysed with computer-assisted pattern analysis using the GelJ v.2.0.
software (Heras et al., 2015). The relatedness of the isolates was compared and dendrograms were constructed by UPGMA and cluster analysis was used to determine the relationships between each isolate. The value of discriminatory power [D) was determined using an online calculator for discriminatory power as reported (Milton et al., 2015).

Data assessment
Data analysis was performed by SPSS Statistics 21.0 (SPSS Inc., Chicago, IL, USA). Chi-square and Fisher's exact two-tailed tests were performed to assess any significant relationship between the Campylobacter prevalence and virulence and antimicrobial resistance properties. Besides, p value < 0.05 was considered statistically significant.
From a statistical seeing, significant differences were found between types of samples and distribution of antimicrobial resistance-encoding genes (p < 0.05). C. jejuni strains harboured a higher and more diverse distribution of antimicrobial resistance-encoding genes than C. coli isolates (p < 0.05).

3.4
Campylobacter virulence characters   TA B L E 4 Antimicrobial resistance-encoding genes distribution among the C. jejuni isolates of examined samples

DISCUSSION
Targeted control of foodborne pathogens usually relies on the identi-

fication of sources and routes of transmission. Poultries can harbour
Campylobacter and represent sources for human campylobacteriosis.
All phases -from primary poultry production to the consumer -play an imperative portion in the Campylobacter transmission (Guirin et al., 2020).
The present survey was done to assess the prevalence, antimicrobial resistance properties, virulence characters and molecular typing of C.   and finally 14.30% and 9.80%, respectively, which confirm our findings of the higher antibiotic resistance of C. jejuni isolates than that of C. coli. However, Giacomelli et al. (2014) reported that the C. jejuni strains isolated from poultry harboured more than 50% susceptibility towards gentamicin, apramycin, streptomycin, cefotaxime, amoxicillin + clavulanic acid, erythromycin, tilmicosin, tylosin, clindamycin and chloramphenicol. Reversely, they showed that majority of C. coli strains were resistant to streptomycin, cephalothin, cefotaxime, ampicillin, nalidixic acid, ciprofloxacin, erythromycin, tilmicosin, tylosin, tetracycline, clindamycin and chloramphenicol. As a result, Giacomelli et al.   (Ghunaim et al., 2015). In keeping with this, some other researchers defined the tendency of susceptible strains to cause more severe infections than resistant ones (Feodoroff et al., 2009). Thus, additional studies should be conducted to explore more in-depth the relationship between the pathogenic traits and the antimicrobial resistance in Campylobacter strains. The high distribution of virulence factors was also reported in surveys conducted in the United States In the final section of the present survey, ERIC-PCR was used for molecular typing of Campylobacter spp. according to findings; the majority of isolates had more than 80% genetic similarities and were classified in the same group. This finding may show their common source and route of transmission into the chicken meat samples. Additionally, high diversity was determined between C. jejuni and C. coli isolated from different raw poultry meat samples. This part of our findings was akin to those reported from South Africa (Igwaran & Okoh, 2020a), Egypt (Ahmed et al., 2015) and India (Milton et al., 2015).

CONCLUSION
In conclusion, virulent and antimicrobial-resistant strains of C. jejuni and C. coli were isolated from chicken, ostrich, turkey, quebec and goose meat samples. Chicken and Turkey meat samples harboured the highest contamination rates. C. jejuni and C. coli isolates harboured a high resistance rate against erythromycin, ampicillin, ciprofloxacin, chloramphenicol and tetracycline, which was accompanied by the high jejuni and C. coli to the human community was also determined. Further investigations should perform to compare the antibiotic resistance pattern, virulence gene profile and ERIC-PCR typing of C. jejuni and C.
coli strains isolated from poultry meat and human being. Additionally, there is a large demand to assess the relationship between virulence characters and antimicrobial resistance properties in C. jejuni and C. coli isolates.

AUTHOR CONTRIBUTIONS
HM and AS carried out the molecular genetic studies, participated in the primers sequence alignment and drafted the manuscript. MH and AS carried out the sampling and culture method. HM and AS participated in the design of the study, performed the statistical analysis and writing the manuscript. All authors read and approved the final manuscript.